CN113820415B - Method for measuring food-borne stimulant in spice based on liquid chromatography-tandem mass spectrometry isotope internal standard method - Google Patents

Abstract

The invention relates to the technical field of detection of food-source stimulants, in particular to a method for measuring the food-source stimulants in spices based on a liquid chromatography-tandem mass spectrometry isotope internal standard method. The method of the invention comprises the following steps: mixing the spice with the internal standard solution to obtain a mixed solution; (2) Adding the extracting solution into the mixed solution, uniformly mixing, and centrifuging to obtain supernatant to obtain spice extracting solution; (3) Purifying the spice extracting solution on an oassimcx solid-phase extraction column, drying the obtained eluent by nitrogen at 39-41 ℃, re-dissolving by a complex solvent, and passing through a membrane to obtain the spice to be detected; (4) And (3) measuring the peak area of the stimulant in the spice to be measured by using a liquid chromatography-tandem mass spectrometry system, and calculating the content of the food-borne stimulant in the spice according to a standard curve. The method can detect endogenous higenamine and Qu Tuokui phenol simultaneously, and is accurate and sensitive.

Description

Method for measuring food-borne stimulant in spice based on liquid chromatography-tandem mass spectrometry isotope internal standard method

Technical Field

The invention relates to the technical field of detection of food-source stimulants, in particular to a method for measuring the food-source stimulants in spices based on a liquid chromatography-tandem mass spectrometry isotope internal standard method.

Background

Food-borne stimulants refer to stimulants derived from food and can be classified into endogenous and exogenous stimulants according to the source. Some foods contain stimulant components which naturally occur or are produced in animals and plants, i.e., endogenous stimulant components such as norlindera root alkaloids and Qu Tuokui phenol are tetrahydroisoquinoline alkaloids which are widely distributed in nature and are mainly distributed in 10 plant groups such as magnoliaceae, camphoraceae, aristolochidae, buttercup, berberidaceae, stephaniae tetrandrae, papaveris and the like.

Only research reports on human urine are seen on Qu Tuokui phenol detection at present. The study report on the detection of norlinderane is more, and the detection matrix comprises biological samples, chinese herbal medicines, plant samples and dietary supplements. Some researches on detection of norlinderane in spice have been started in recent years, and mainly comprise liquid chromatography-electrochemical method and liquid chromatography-tandem mass spectrometry. Although the liquid chromatography-electrochemical method can eliminate the interference of most of non-electroactive substances in complex samples, the sensitivity is poor, and the monitoring requirement cannot be met. The liquid chromatography-tandem mass spectrometry adopts a matrix standard curve external standard method to quantify the compound, and norlinderane is an endogenous drug, so that a blank matrix standard curve is difficult to prepare for some positive matrix samples, and accurate quantification of the compound cannot be realized. There are few liquid chromatography-tandem mass spectrometry methods for determining compounds by using an internal standard method, but the purification mode in the method can absorb the norlinderane to a certain extent, which is very easy to cause inaccurate detection data. The prior art does not find a sensitive and accurate method for simultaneously detecting norlinderamine and Qu Tuokui phenol in spice samples.

Disclosure of Invention

The invention aims to provide a sensitive and accurate method for simultaneously detecting norlinderamine and Qu Tuokui phenol in a spice sample.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for measuring food-source stimulants in spices based on a liquid chromatography-tandem mass spectrometry isotope internal standard method, which comprises the following steps:

(1) Mixing the spice with the internal standard solution to obtain a mixed solution;

(2) Adding the extracting solution into the mixed solution, uniformly mixing, and centrifuging to obtain supernatant to obtain spice extracting solution;

(3) Purifying the spice extracting solution on an Oasis MCX solid phase extraction column, drying the obtained eluent by nitrogen at 39-41 ℃, re-dissolving by a complex solvent, and passing through a membrane to obtain the spice to be detected;

(4) And (3) measuring the peak area of the stimulant in the spice to be measured by using a liquid chromatography-tandem mass spectrometry system, and calculating the content of the food-borne stimulant in the spice according to a standard curve.

Preferably, the mass volume ratio of the spice to the internal standard solution is 1 g:49-51 mu L;

the internal standard solution is a mixed solution of D4-norlinderane and D9-Qu Tuokui phenol;

the mass concentration of the D4-norlinderane is 10ng/mL;

the mass concentration of the D9-Qu Tuokui phenol is 10ng/mL.

Preferably, the volume-mass ratio of the addition amount of the extracting solution to the spice is 9.5-10.5 mL/1 g;

the extracting solution is 0.4-0.6% formic acid ethanol solution;

the volume ratio of 0.4-0.6% formic acid to ethanol in 0.4-0.6% formic acid ethanol solution is 1:198-200.

Preferably, the mode of mixing is homogeneous mixing;

the homogenizing rotating speed is 9000-11000 r/min;

homogenizing for 0.8-1.2 min;

the rotational speed of the centrifugation is 9000-11000 r/min;

the centrifugation time is 4-6 min.

Preferably, the eluent in the elution is 4.5-5.5% ammonia water methanol solution;

the volume ratio of the 4.5 to 5.5 percent ammonia water to the methanol in the 4.5 to 5.5 percent ammonia water methanol solution is 1:18 to 20;

the volume-mass ratio of the addition of the eluent to the spice during elution is 1.8-2.2 mL/1 g.

Preferably, the cosolvent is a mixed solution of formic acid aqueous solution and methanol;

the mass concentration of the aqueous solution of the formic acid is 0.09-0.11%;

the volume ratio of the formic acid aqueous solution to the methanol is 3.5-4.5:1;

the volume mass ratio of the addition amount of the complex solvent to the spice is 0.45-0.55 mL:1g;

the membrane is an organic filter membrane;

the pore size of the organic filter was 0.22. Mu.m.

Preferably, the chromatographic conditions in the liquid chromatography-tandem mass spectrometry system are: the column was Waters ACQUITY UPLC BEH C with a diameter of 2.1X100 mm and a diameter of 1.7. Mu.m ₁₈ A chromatographic column; mobile phase A is formic acid water solution with mass concentration of 0.1%; mobile phase B is methanol solution; the flow rate is 0.4mL/min; the column temperature is 35 ℃; the sample injection amount is 5 mu L;

the gradient elution program is 0-0.5 min,10% of mobile phase B;

0.5 to 3.5min,10 to 90 percent of mobile phase B;

3.5 to 5min,90% of mobile phase B;

5 to 5.01min,90 to 10 percent of mobile phase B;

5.01-7 min,10% mobile phase B.

Preferably, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry system are: electrospray ionization source ESI, reaction monitoring mode is MRM; the temperature of the ion source is 350 ℃; the capillary temperature was 250 ℃; the temperature of the heating module is 350 ℃; nitrogen flow rate: 3.0L/min, drying gas flow rate: 10.0L/min, heating air flow rate: 10.0L/min.

Preferably, the stimulant is a higenamine and/or a tritoquinol.

Preferably, the standard curve of the higenamine is y=3.19066x+0.181625, r= 0.99989;

the standard curve for Qu Tuokui phenol is y=0.906370x+0.0299261, r= 0.99994;

x represents: the ratio of the concentration of the stimulant contained in the sample to the concentration of the corresponding stimulant contained in the internal standard solution;

y represents: the ratio of the peak area of the stimulant contained in the sample to the peak area of the corresponding stimulant contained in the internal standard solution.

The invention provides a method for measuring food-source stimulants in spice based on a liquid chromatography-tandem mass spectrometry isotope internal standard method. The method provided by the invention applies Oasis MCX solid phase extraction column purification, and establishes a detection method of Qu Tuokui phenol in the spice by using an ultra-high performance liquid chromatography-quadrupole tandem mass spectrometry method. The experiment optimizes the pretreatment method and the chromatographic mass spectrometry condition respectively, and the content of the norlinderamine and the Qu Tuokui phenol in the spice can be quantitatively determined by adopting a multi-reaction monitoring mode for detection and an isotope internal standard method. The method has the advantages of high sensitivity, universality, simplicity and convenience in operation and the like, and can provide effective technical support for food guarantee work of sports events.

Drawings

FIG. 1 shows the selection of different extracts.

Fig. 2 is a scanning pattern of norlinderane ions.

Fig. 3 is a scan of a tritoquinol ion.

Figure 4 is the effect of mobile phase composition on norlinderamine and Qu Tuokui phenol.

FIG. 5 is a MRM chromatogram (wherein the left panel is a blank octagon sample and the right panel is an octagon sample with 2. Mu.g/kg of norlinderamine added).

FIG. 6 is a MRM chromatogram (wherein the left panel is a blank octagon sample and the right panel is a octagon sample with 2. Mu.g/kg Qu Tuokui phenol added).

Figure 7 shows the matrix effect of norlinderamine and trazoquinol.

Detailed Description

The chemical structural formula of the norlinderane is as follows:

the chemical structural formula of the Qu Tuokui phenol provided by the invention is as follows:

the technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Sources of instrumentation used in embodiments of the present invention:

UPLC 8050 ultra-high performance liquid chromatography tandem mass spectrometer was purchased from Shimadzu corporation;

model number of centrifuge was 3K-15, purchased from Sigma Co., USA;

the model of the homogenizer was PT2100, available from KINEMATICA company, switzerland;

the nitrogen blower model is N-EVAP112, available from organization company of America;

vortex mixers are available from us Scientific Industries company;

Milli-Q purification System was purchased from Millipore company, USA;

WatersACQUITYUPLC BEH C ₁₈ chromatographic column 2.1X100 mm,1.7 μm, oasis MCX solid phase extraction column 60mg,3mL and Oasis PRiME HLB solid phase extraction column 60mg,3mL were all purchased from Waters, USA;

60mg of CleanertPCX solid phase extraction column, 3mL, was purchased from Agela company, U.S.A.

Sources of reagents used in the examples of the present invention:

acetonitrile, methanol and ethanol, of chromatographic purity, available from Merck, germany;

formic acid purity was chromatographic purity, purchased from Fluka corporation, germany;

the water is Milli-Q high-purity water;

the purity of the norlinderane is 98.0%, the concentration of Qu Tuokui phenol is 100 mug/mL, the concentration of D4-norlinderane is 100 mug/mL, and the concentration of D9-Qu Tuokui phenol is 100 mug/mL, all of which are purchased from Tianjin Alta technologies Co., ltd;

star anise, cumin, murraya paniculata, fennel, star anise, dried chilli, herb of common nutmeg, bergamot, hawthorn, cassia twig, kaempferia galanga, long pepper, cardamon, angelica dahurica, gardenia, pricklyash peel, black pepper, white pepper, chive, bay leaf, clove, galangal, citronella, dried orange peel, cinnamon, rapeseed, leek seed, poria cocos, gorgon fruit and medlar are all randomly extracted from the market.

The configuration method of the internal standard solution in the embodiment of the invention comprises the following steps:

(1) 1mL of each of D4-norlinderane and D9-Qu Tuo quinol standard is removed, and methanol is used for preparing a mixed internal standard intermediate solution with the concentration of 1.0 mug/mL;

(2) And preparing 10ng/mL of the mixed internal standard intermediate solution by using 0.1% formic acid-methanol with the volume ratio of 8:2, so as to obtain the internal standard solution.

0.1% formic acid is an aqueous solution of formic acid having a volume concentration of 0.1%.

The preparation method of the mixed standard solution of the norlinderane and the Qu Tuokui phenol in the embodiment of the invention comprises the following steps:

(1) Preparing norlinderane into 100 mug/mL norlinderane solution by methanol;

(2) Respectively transferring 100 μg/mL of the norcombined spicebush root alkali solution and Qu Tuokui phenol 1mL, dissolving with methanol, and preparing into mixed standard intermediate with the mass concentration of 1.0 μg/mL;

(3) The mixed standard intermediate solution is prepared into a mixed standard solution with the mass concentration of 0.04,0.1,0.5,2.0, 10.0 and 50.0ng/mL by using 0.1 percent formic acid-methanol with the volume ratio of 8:2.

Other parameters of mass spectrometry analysis of norlinderane and Qu Tuokui phenol in the examples of the present invention are shown in table 1, and the x in table 1 is the quantitative ion.

Table 1 mass spectrometry parameters of norlinderane and Qu Tuokui phenol

Example 1

Accurately weigh 2g of the sample into a 50mL plastic centrifuge tube. 100 mu L of internal standard solution is added and mixed by vortex. Adding 20mL of the extract, and homogenizing for 1min at 10000r/min on a homogenizer. Centrifuging at 10000r/min for 5min, and collecting appropriate amount of supernatant, and filtering with a membrane to obtain sample extractive solution.

The Oasis MCX solid phase extraction column is activated by 3mL of methanol and 3mL of water respectively, 2mL of the sample extracting solution is put on the solid phase extraction column, and after the sample solution flows out, the sample solution is leached by 3mL of water and 3mL of methanol in sequence and pressed to dryness. Finally, 4mL of 5% ammonia water and methanol are used for eluting, the eluent is received and is blown to be dry by nitrogen at 40 ℃, 1mL of complex solvent is used for vortex dissolution of residues, and the residues are filtered through an organic filter membrane of 0.22 mu m to obtain a sample to be tested.

Chromatographic conditions: watersACQUITYUPLC BEH C ₁₈ 2.1X100 mm,1.7 μm column; mobile phase a and mobile phase B; flow rate: 0.4mL/min; column temperature: 35 ℃; sample injection amount: 5. Mu.L; gradient elution procedure: 0-0.5 min,10% mobile phase B;0.5 to 3.5min,10 to 90 percent of mobile phase B;3.5 to 5min,90% of mobile phase B;5 to 5.01min,90 to 10 percent of mobile phase B; 5.01-7 min,10% mobile phase B.

Mass spectrometry conditions: electrospray ionization source ESI (+), multi-reaction monitoring (MRM) mode. The ion source temperature was 350 ℃, the capillary temperature was 250 ℃, the heating module temperature was 350 ℃, the nitrogen flow rate: 3.0L/min, drying gas flow rate: 10.0L/min, heating air flow rate: 10.0L/min. Other parameter settings are shown in table 1.

(1) Optimization of the extract

The selection of the extraction solution aims at extracting more target compounds and fewer impurities, the recovery rate of the norlinderane and Qu Tuokui phenol and the response value of the matrix standard solution are used as judgment standards, and 100ng/mL of mixed standard solution of the norlinderane and Qu Tuokui phenol is added into a blank sample to be used as a simulation sample for investigation.

The blank sample of the experiment selects star anise, cumin, fennel, star anise, capsicum and leek seeds, and compares the influence of three solutions of 80% methanol aqueous solution, 0.5% formic acid-ethanol solution and 0.5mM sodium acetate solution pH=5.2 as extracting solutions on the recovery rate of the norlinderane and Qu Tuokui phenol. The results are shown in FIG. 1.

FIG. 1 shows that 0.5mM sodium acetate has poor extraction effect, the recovery rate is between 50 and 60 percent, the extraction effect of the 80 percent methanol solution and the extraction effect of the 0.5 percent formic acid ethanol are equivalent, the recovery rate is over 80 percent, but the extraction color of the 80 percent methanol solution is darker, and in order to avoid the difficulty in subsequent purification, the 0.5 percent formic acid ethanol is selected as the extraction solvent in the study.

(2) Purification column selection

The spice seasoning has complex and various matrix components, and a solid phase extraction purification mode is needed to reduce background interference and reduce matrix effect. Because the norlinderane and Qu Tuokui phenol have the characteristics of easily forming cations under acidic conditions and easily becoming molecules under alkaline conditions.

The experiment compares the purification effects of six matrixes including star anise, cumin, fennel, large material, chilli and Chinese chive seeds by using the mixed cation exchange columns Oasis MCX, oasis priME HLB and PCX solid phase extraction small column. The results are shown in Table 2.

Table 2 shows that the cation exchange column Oasis MCX has the best purification effect and stable recovery rate.

TABLE 2 influence of different solid phase extraction columns on recovery of norlinderane and Qu Tuokui phenol

(3) Optimization of mass spectrometry conditions

And injecting 1mg/L standard substance mixed solution into a mass spectrum system in a mode of not connecting an analysis column, wherein the sample injection amount is 1 mu L. And (3) performing total scanning of parent ions in a positive ion mode, determining molecular ion peaks, performing total scanning by taking the molecular ion peaks of the object to be detected as parent ions, selecting two fragments with the highest response values as characteristic ions, optimizing out the optimal collision energy CE and Q1 and Q3 voltages, and finally determining the ion pairs and the collision energy of multi-reaction monitoring, wherein the established mass spectrum parameters are shown in a table 1. The sub-ion scan of the norlinderane and Qu Tuokui phenol and their internal standard are shown in figures 2-3.

(4) Chromatographic condition optimization

Experimental selection Waters ACQUITY UPLC BEH C ₁₈ The chromatographic column was 2.1X100 mm,1.7 μm, and the sensitivity and peak profile of norlinderane and Qu Tuokui phenol were compared with those of 0.1% formic acid-acetonitrile, water-methanol, 0.01% formic acid-methanol, 0.05% formic acid-methanol, 0.1% formic acid-methanol as mobile phase, by separating norlinderane and Qu Tuokui phenol. The results are shown in FIG. 4.

Figure 4 shows that norlinderane and Qu Tuokui phenol were highly sensitive in 0.1% formic acid-methanol, but peak-type was poor. Formic acid with a certain content is added into the water phase, so that the peak type can be effectively improved. Fig. 4 shows that 0.05% formic acid-methanol as mobile phase can meet the detection requirement of sensitivity, and the peak shape is sharp.

And then, through experimental optimization, 0.1% formic acid-methanol constant volume with the volume ratio of 8:2 is adopted, so that the complete dissolution of the compound can be ensured, and the good peak shape can be ensured.

The MRM chromatogram of adding 2. Mu.g/kg of norlinderane and Qu Tuokui phenol to an octagon blank was observed using this chromatographic condition. The results are shown in FIGS. 5 to 6.

(5) Matrix effect

In the experiment, star anise, cumin, fennel, star anise, capsicum and Chinese chive seed blank samples are selected as matrixes, and the blank samples are processed by adopting an optimized sample processing method to respectively prepare a matrix matching standard curve and a solvent standard curve. Matrix effects were calculated from me= [ matrix matching standard curve slope/solvent standard curve slope-1 ] ×100%. The matrix effect pattern of each matrix is shown in FIG. 7.

Figure 7 shows that matrix response of 5 matrices is matrix weakening, |me| is 0.38-0.75 and the norlindera base quality effect is stronger than that of trastuquinol. In order to ensure the accuracy of the quantitative result, the quantitative result is corrected by adopting an isotope internal standard method to eliminate the interference of the basic norlindera root alkali on the quantitative result.

Example 2

(1) Linear relationship, detection limit and quantification limit of method

The mixed standard solution of 0.04,0.1,0.5,2.0, 10.0 and 50.0ng/mL and the internal standard solution of 10ng/mL are taken and analyzed in order of concentration from low to high. The ratio of the concentration of the stimulant corresponding to the standard solution to the concentration of the corresponding stimulant contained in the internal standard solution is taken as an abscissa; drawing a standard curve by taking the ratio of the peak area of the stimulant corresponding to the standard solution to the peak area of the stimulant contained in the corresponding isotope internal standard solution as an ordinate to obtain a linear equation and a correlation coefficient R ² 。

A low-concentration standard solution is added into star anise, cumin, fennel, star anise, capsicum and Chinese chive seed blank matrix samples, the sample treatment method optimized in the embodiment 1 and the chromatographic mass spectrometry conditions are used for measurement, and the detection limit LOD is 0.3 mug/kg and the quantitative limit LOQ is 1.0 mug/kg according to the signal to noise ratio S/N being more than or equal to 3 and S/N being more than or equal to 10. Qu Tuokui phenols have good linear relationship in the linear range of 0.04-50 ng/mL in each matrix, and the linear equation of the norlinderane is Y=3.19066X+0.181625, and the correlation coefficient line 0.99989; the Qu Tuokui phenol linear equation is y=0.906370x+0.0299261, and the correlation coefficient is 0.99994.

(2) Recovery and precision of the method

The recovery rate and precision of 6 spice seasonings without norlinderamine and Qu Tuokui phenol, which are selected from star anise, cumin, fennel, star anise, capsicum and leek seeds, are verified by a standard adding recovery method, and an addition recovery experiment is carried out according to the optimized treatment method and chromatographic mass spectrometry conditions of the example 1 under the mass concentration of 1.0, 2.0, 10 and 100 mug/kg for 4 respectively, each level is measured for 6 times, and the average recovery rate and the relative standard deviation are shown in Table 3. As is clear from Table 3, the recovery rates of the methods were all between 74.5 and 98.8%, and the relative standard deviations were between 3.41 and 11.04%.

Table 3 method of standard recovery and relative standard deviation (n=6)

Example 3

31 spice samples of star anise, cumin, murraya paniculata, fennel, star anise, dried chilli, row grass, nutmeg, bergamot, hawthorn, cassia twig, kaempferia galanga, long pepper, cardamon, angelica dahurica, gardenia, pepper, black pepper, white pepper, chive, bay leaves, clove, galangal, lemongrass, dried orange peel, cinnamon, rapeseed, leek seed, tuckahoe, gorgon fruit and medlar purchased in the market were measured by adopting the optimized treatment method and the optimized chromatographic mass spectrometry method in example 1, and Qu Tuokui phenol was not detected. Wherein the content of norlinderamine is shown in Table 4.

Table 4 content of norlinderane in actual sample (n=3)

From the above examples, the present invention provides a method for determining food-borne stimulants in spices based on liquid chromatography-tandem mass spectrometry isotope internal standard method. The method provided by the invention applies Oasis MCX solid phase extraction column purification, and establishes a detection method of Qu Tuokui phenol in the spice by using an ultra-high performance liquid chromatography-quadrupole tandem mass spectrometry method. The experiment optimizes the pretreatment method and the chromatographic mass spectrometry condition respectively, and adopts a multi-reaction monitoring mode for detection, and the isotope internal standard method can quantitatively determine the content of the norlinderamine and the Qu Tuokui phenol in the spice. The method has the advantages of high sensitivity, universality, simplicity and convenience in operation and the like, and can provide effective technical support for food guarantee work of sports events.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (6)

1. The method for determining the food-borne stimulant in the spice based on the liquid chromatography-tandem mass spectrometry isotope internal standard method is characterized by comprising the following steps of:

(1) Mixing the spice with the internal standard solution to obtain a mixed solution;

(2) Adding the extracting solution into the mixed solution, uniformly mixing, and centrifuging to obtain supernatant to obtain spice extracting solution;

(3) Purifying the spice extracting solution on an Oasis MCX solid phase extraction column, drying the obtained eluent by nitrogen at 39-41 ℃, re-dissolving by a complex solvent, and passing through a membrane to obtain the spice to be detected;

(4) Measuring the peak area of the stimulant in the spice to be measured by using a liquid chromatography-tandem mass spectrometry system, and calculating to obtain the content of the food-borne stimulant in the spice according to a standard curve;

the extracting solution is 0.4-0.6% formic acid-ethanol solution, the volume ratio of 0.4-0.6% formic acid to ethanol in the 0.4-0.6% formic acid-ethanol solution is 1:198-200, and the adding amount of the extracting solution to the volume-mass ratio of the spice is 9.5-10.5 mL:1g;

the eluent in the elution is 4.5-5.5% ammonia water methanol solution, the volume ratio of 4.5-5.5% ammonia water to methanol in the 4.5-5.5% ammonia water methanol solution is 1:18-20, and the volume mass ratio of the addition of the eluent to the spice in the elution is 1.8-2.2 mL:1g;

the complex solvent is a mixed solution of formic acid aqueous solution and methanol, the mass concentration of the formic acid aqueous solution is 0.09-0.11%, the volume ratio of the formic acid aqueous solution to the methanol is 3.5-4.5:1, and the volume mass ratio of the addition amount of the complex solvent to the spice is 0.45-0.55 mL:1g;

the membrane is an organic filter membrane;

the pore diameter of the organic filter membrane is 0.22 mu m;

the chromatographic column is a Waters ACQUITY UPLC BEH C chromatographic column with the diameter of 2.1 multiplied by 100mm and the diameter of 1.7 mu m; mobile phase A is formic acid water solution with mass concentration of 0.1%; mobile phase B is methanol solution;

the gradient elution program is 0-0.5 min,10% of mobile phase B;

0.5 to 3.5min,10 to 90 percent of mobile phase B;

3.5 to 5min,90% of mobile phase B;

5 to 5.01min,90 to 10 percent of mobile phase B;

5.01-7 min,10% of mobile phase B;

the stimulant is norlinderamine and Qu Tuokui phenol.

2. The method according to claim 1, wherein the mass-to-volume ratio of the spice to the internal standard solution is 1 g:49-51 μl;

the internal standard solution is a mixed solution of D4-norlinderane and D9-Qu Tuokui phenol;

the mass concentration of the D4-norlinderane is 10ng/mL;

the mass concentration of the D9-Qu Tuokui phenol is 10ng/mL.

3. The method according to claim 2, wherein the mixing is by homogenization;

the homogenizing rotating speed is 9000-11000 r/min;

homogenizing for 0.8-1.2 min;

the rotational speed of the centrifugation is 9000-11000 r/min;

the centrifugation time is 4-6 min.

4. A method according to claim 3, wherein the chromatographic conditions in the liquid chromatography-tandem mass spectrometry system are: the flow rate is 0.4mL/min; the column temperature is 35 ℃; the sample injection amount is 5 mu L.

5. The method of claim 4, wherein the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry system are: electrospray ionization source ESI, reaction monitoring mode is MRM; the temperature of the ion source is 350 ℃; the capillary temperature was 250 ℃; the temperature of the heating module is 350 ℃; nitrogen flow rate: 3.0L/min, drying gas flow rate: 10.0L/min, heating air flow rate: 10.0L/min.

6. The method of claim 5, wherein the standard curve for norlinderane is y=3.19066x+0.181625, r= 0.99989;

the standard curve for Qu Tuokui phenol is y=0.906370x+0.0299261, r= 0.99994;

x represents: the ratio of the concentration of the stimulant contained in the sample to the concentration of the corresponding stimulant contained in the internal standard solution;

y represents: the ratio of the peak area of the stimulant contained in the sample to the peak area of the corresponding stimulant contained in the internal standard solution.